Study on the enantiomerization of synephrine by chiral high-performance liquid chromatography

Synephrine, the active phenethylamine alkaloid of Citrus aurantium L., is a biologically active compound that has effects on human metabolism that could help to reduce fat mass in obese people, since it stimulates lipolysis, raises the metabolic rate and promotes the oxidation of fat through increased thermogenesis. In the light of this, C. aurantium extracts are commonly used in the formulation of phytotherapic products employed in the treatment of obesity. It is well known that synephrine is a chiral compound and its enantiomers have shown different pharmacological activity on alpha- and beta-adrenoreceptors. In particular, R-(−)-synephrine is from 1 to 2 orders of magnitude more active than its S-(+)-counterpart. R-(−)-Synephrine has been isolated and identified in Citrus fruits. However, a certain amount of S-(+)-synephrine has been detected in C. aurantium dry extracts and dietary supplements (1). The presence of S-(+)-synephrine in C. aurantium natural products has been attributed to a possible enantiomerization of R-(-)-synephrine during the industrial production of the fruit extracts, using a high temperature and a long period of refluxing. To evaluate the enantiomerization kinetic parameters of R-(-)-synephrine, an off-column HPLC method based on a chiral stationary phase (CSP) with cellobiohydrolase as the chiral selector (Chiral-CBH) was developed. Analyses were carried out on a Chiral-CBH column (100 × 4.0 mm i.d., 5 um), with a mobile phase consisting of 2-propanol (5%, w/w) in sodium phosphate buffer (pH 6.0; 10 mM) and disodium EDTA (50 uM). The flow rate was 0.8 mL/min. The column was thermostatted at 20°C. Detection was set at 225 nm. The individual enantiomers of the studied compound were isolated by fractional crystallization of the diastereomeric salts and subsequent ion-exchange. The rate constants and the free energy barriers of enantiomerization were determined in different solvents and buffer solutions at pH 1-11. The results generated by the off-line method were used to determine the influence of solvents and pH values on the enantiomerization rate of (+) and (-)-synephrine and to gain further insight into the enantiomerization mechanism of chiral phenethylamine type alkaloids in relation to the pKa values.